Target:5-nucleotidase SurE RefSeq: CT218 Protein ID: NP_219722.1 Organism: Chlamydia trachomatis (D/UW-3/CX) Etiologic Risk Group: Risk Group 2 Background/Disease Information: Trachoma is an infectious disease caused by Chlamydia trachomatis, and is theleading cause of infectious blindness in the world. Trachoma causes a roughening of the inner surface of the eyelids, eye discharge, eye scarring, ingrown eyelashes, and other painful symptoms. WHO Guidelines recommend the use of antibiotics such as Azithromycin or tetracycline to treat the infection. This affliction is currently classified as a neglected tropical disease [1]. Essentiality of this protein: "5-nucleotidases (members of EC 3.1.3.5 and EC 3.1.3.6) dephosphorylate non-cyclic nucleoside monophosphates to nucleosides and inorganic phosphate" [2] "DNA and RNA synthesis requires a continuous and balanced supply of the four deoxyribonucleotides and four ribonucleotides. A network of ... enzymes regulates the size of each pool with the enzymes ribonucleotide reductase, nucleoside kinases, and nucleotidases playing the main roles." [3]. By inhibiting the function of this enzyme, the regulation of nucleotides and phosphorus is depleted, leading to the organism's inability to continue essential cell functions. Complex of proteins: No DruggableTarget: There are several known inhibitors, indicating that it may be possible to drug this target using manufactured compounds [2]. EC#:3.1.3.5 Link to BRENDA EC# page:http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.5 Enzyme Assay information: "Purified E. coli proteins were screened for phosphatase activity using general phosphatase substrate p-nitrophenyl phosphate (pNPP) in 96-well microplates. The reaction mixture (0.2 ml) contained 50 mM HEPES-K buffer (pH 7.5), 5 mM MgCl2, 0.5 mM MnCl2, 0.5 mM NiCl2, 40 mM pNPP, 0.1–1.0 g of purified protein. After addition of protein samples, the plates were incubated for 1–3 h at 37 °C before taking the reading at 410 nm" [2].
Cost and Quantity of Substrate Reagents and Supplier: 50mM HEPES-K Buffer (Sigma): $38.70/25g 5mM MgCl2 (Sigma): $43.60/100g 0.5mM MnCl2 (Sigma): $45.50/10g 0.5mM NiCl2 (Sigma): $36.00/50g 40mM pNPP (Sigma): $79.60/1g 0.1-1.0g 5nucleotidase SurE (Ontario Centre for Structural Proteomics): No Price Given [2] 3'-CMP Cytidine-3'-monophosphate (Santa Cruz Biotechnology, Inc.): $98/1g Structure Available: Homology Model Closest PDB Entry: 1J9J Homology Model Option: Query Coverage: 65% Max % Identities: 37% % Positives: 55% Chain Used for Homology: Chain A
Current Inhibitors: pyrimidine nucleotide, nucleoside analogs, dTTP, dGTP, dCTP, dATP [2]. Expression Information: Expressed in E.coli DH5α [2]. Purification Method: "To prepare protein samples for screening, recombinant proteins containing an N-terminal His6 fusion tag were expressed in 1-liter E. coli BL21(DE3) cultures and affinity-purified on nickel-nitrilotriacetic acid resin (Qiagen) as previously described (17). Briefly, cell lysates were loaded onto small (1.5 x 4 cm) nickel columns equilibrated with loading buffer (50 mM HEPES-K, pH 7.5; 0.5 M NaCl; 5 mM imidazole), washed with 10 volumes of washing buffer (loading buffer containing 30 mM imidazole), and eluted with 250 mM imidazole in loading buffer" [2].
Length of protein in Amino Acids: 283 Molecular Weight of protein in kiloDaltons: 31544.8 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 31525 M-1 cm-1
NOT NECESSARY FOR SPRING Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
RefSeq: CT218
Protein ID: NP_219722.1
Organism: Chlamydia trachomatis (D/UW-3/CX)
Etiologic Risk Group: Risk Group 2
Background/Disease Information: Trachoma is an infectious disease caused by Chlamydia trachomatis, and is theleading cause of infectious blindness in the world. Trachoma causes a roughening of the inner surface of the eyelids, eye discharge, eye scarring, ingrown eyelashes, and other painful symptoms. WHO Guidelines recommend the use of antibiotics such as Azithromycin or tetracycline to treat the infection. This affliction is currently classified as a neglected tropical disease [1].
Essentiality of this protein: "5-nucleotidases (members of EC 3.1.3.5 and EC 3.1.3.6) dephosphorylate non-cyclic nucleoside monophosphates to nucleosides and inorganic phosphate" [2] "DNA and RNA synthesis requires a continuous and balanced supply of the four deoxyribonucleotides and four ribonucleotides. A network of ... enzymes regulates the size of each pool with the enzymes ribonucleotide reductase, nucleoside kinases, and nucleotidases playing the main roles." [3]. By inhibiting the function of this enzyme, the regulation of nucleotides and phosphorus is depleted, leading to the organism's inability to continue essential cell functions.
Complex of proteins: No
Druggable Target: There are several known inhibitors, indicating that it may be possible to drug this target using manufactured compounds [2].
EC#: 3.1.3.5
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.5
Enzyme Assay information: "Purified E. coli proteins were screened for phosphatase activity using general phosphatase substrate p-nitrophenyl phosphate (pNPP) in 96-well microplates. The reaction mixture (0.2 ml) contained 50 mM HEPES-K buffer (pH 7.5), 5 mM MgCl2, 0.5 mM MnCl2, 0.5 mM NiCl2, 40 mM pNPP, 0.1–1.0 g of purified protein. After addition of protein samples, the plates were incubated for 1–3 h at 37 °C before taking the reading at 410 nm" [2].
Link to Sigma page for assay or assay reagents (substrates):
HEPES-K Salt
MgCl2
MnCl2
NiCl2
PPnPP
5nucleotidase SurE
Cost and Quantity of Substrate Reagents and Supplier:
50mM HEPES-K Buffer (Sigma): $38.70/25g
5mM MgCl2 (Sigma): $43.60/100g
0.5mM MnCl2 (Sigma): $45.50/10g
0.5mM NiCl2 (Sigma): $36.00/50g
40mM pNPP (Sigma): $79.60/1g
0.1-1.0g 5nucleotidase SurE (Ontario Centre for Structural Proteomics): No Price Given [2]
3'-CMP Cytidine-3'-monophosphate (Santa Cruz Biotechnology, Inc.): $98/1g
Structure Available: Homology Model
Closest PDB Entry: 1J9J
Homology Model Option:
Query Coverage: 65%
Max % Identities: 37%
% Positives: 55%
Chain Used for Homology: Chain A
Current Inhibitors: pyrimidine nucleotide, nucleoside analogs, dTTP, dGTP, dCTP, dATP [2].
Expression Information: Expressed in E.coli DH5α [2].
Purification Method: "To prepare protein samples for screening, recombinant proteins containing an N-terminal His6 fusion tag were expressed in 1-liter E. coli BL21(DE3) cultures and affinity-purified on nickel-nitrilotriacetic acid resin (Qiagen) as previously described (17). Briefly, cell lysates were loaded onto small (1.5 x 4 cm) nickel columns equilibrated with loading buffer (50 mM HEPES-K, pH 7.5; 0.5 M NaCl; 5 mM imidazole), washed with 10 volumes of washing buffer (loading buffer containing 30 mM imidazole), and eluted with 250 mM imidazole in loading buffer" [2].
Image of Protein:
Amino Acid Sequence:
MTETRRLRILITNDDGIKAKGISLLISLLREADFADLYVVAPLEEQSGRSMAFSLVEPTA
LEPFDYPQRVQEAWAVTGTPVDCVKLAIGELFKENALDLILSGINNGKNSGRCLYYSATV
GAIREANLHGIPAIALSQSENIAFFQKAHMASLIRSLCEFTVAYKHTDPLGLNVNFPAST
DDSPWKGIRFTLSGNEFLFGIPRLVRTEGNRRYYTLYDMRDKVSEEFSEEYLALANNYIS
AAPLVSKNTPRATLSEEELAFLKDSFEQSVLWKASLNLEEDLA
Length of protein in Amino Acids: 283
Molecular Weight of protein in kiloDaltons: 31544.8 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 31525 M-1 cm-1
TMpred graph Image:
CDS Gene Sequence:
ATGACAGAGACACGACGACTTCGCATTCTTATTACCAATGATGACGGTATCAAAGCCAAA GGAATCAGTCTGCTGATTTCTCTACTCCGTGAAGCTGACTTCGCCGATCTTTATGTAGTA GCCCCTTTAGAAGAACAGTCTGGGAGAAGTATGGCTTTCTCATTAGTAGAGCCAACTGCC CTTGAGCCTTTTGATTATCCTCAAAGAGTCCAAGAAGCTTGGGCTGTCACCGGCACTCCT GTTGACTGTGTGAAATTAGCTATAGGAGAACTCTTTAAAGAGAACGCTCTGGATCTTATT TTATCGGGTATTAATAATGGGAAAAATTCCGGACGCTGCCTTTATTACTCTGCCACTGTA GGAGCTATAAGAGAAGCGAATCTCCATGGAATTCCTGCGATAGCTCTTTCTCAAAGTGAG AATATCGCTTTTTTCCAAAAAGCTCATATGGCCTCCCTAATTCGTTCCTTATGCGAGTTT ACCGTTGCCTATAAGCACACCGATCCCTTAGGACTTAATGTAAATTTCCCTGCTAGCACG GACGATTCTCCATGGAAAGGAATTCGTTTCACACTTTCTGGAAATGAATTCTTATTCGGT ATCCCAAGATTAGTCCGTACGGAAGGAAATCGTCGTTACTATACGCTCTATGATATGCGA GATAAAGTATCCGAAGAGTTCTCTGAAGAGTATCTTGCTTTAGCAAATAACTATATTAGT GCGGCTCCACTAGTTTCTAAAAATACCCCTCGAGCAACCCTTTCCGAAGAAGAACTAGCT TTCCTAAAAGACTCTTTCGAACAATCTGTGTTGTGGAAAGCTTCTTTAAACTTAGAAGAA
GATCTGGCTTAG
GC% Content for gene: 42%
CDS Gene Sequence:
ATGACCGAGA CTCGTCGACT TCGTATCCTA ATTACAAATG ATGATGGGAT TAAAGCTAAG GGCATTTCTC TTCTAATTTC CTTACTCCGC GAAGCGGATT
TTGCTGATTT GTATGTGGTC GCCCCACTTG AGGAACAATC TGGACGATCT ATGGCTTTTT CTTTAGTAGA ACCTACAGCA CTAGAACCTT TCGACTATCC
ACAAAGAGTA CAAGAGGCTT GGGCTGTTAC CGGAACACCT GTGGACTGTG TTAAATTAGC TATCGGGGAG TTATTTAAGG AAAATGCCCT CGACCTTATT
CTTTCTGGTA TTAACAATGG AAAAAACTCA GGTCGTTGCT TGTATTATTC TGCCACCGTC GGAGCTATTC GTGAAGCAAA TCTTCACGGA ATCCCTGCAA
TCGCACTTTC TCAGTCTGAG AACATCGCCT TTTTCCAGAA AGCCCACATG GCATCTTTGA TTCGATCTTT GTGCGAATTC ACCGTGGCTT ATAAACATAC
TGACCCTTTA GGGTTAAATG TCAATTTTCC AGCTTCTACT GACGATTCTC CATGGAAAGG TATCCGCTTC ACATTATCCG GAAACGAATT CTTATTTGGT
ATTCCTAGAT TAGTTCGCAC AGAAGGGAAC CGACGTTATT ACACCCTATA TGACATGCGT GATAAAGTCT CCGAGGAATT TTCAGAAGAA TATCTAGCAT
TGGCTAATAA CTATATATCT GCTGCTCCTC TAGTATCAAA AAACACACCA AGAGCAACAC TTTCTGAGGA AGAATTGGCG TTCTTAAAAG ATTCCTTCGA
GCAAAGCGTT CTTTGGAAGG CTTCTTTGAA TCTCGAAGAA GACCTCGCCT AA
GC% Content for Gene: 42.1%
NOT NECESSARY FOR SPRING
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
References:
[1] http://www.who.int/blindness/causes/trachoma/en/index.html
[2] http://www.jbc.org/content/279/52/54687.full.pdf
[3] http://www.jbc.org/content/278/47/46195.full.pdf
[4] http://patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=2754